The present invention relates to a method for the quantitative determination of cholesterol in low density lipoproteins (LDL) (hereinafter referred to as LDL cholesterol), which is important in the field of clinical diagnosis, and a reagent for use in the method. The present invention also relates to a method for the continuous fractional determination of cholesterol in high density lipoproteins (HDL) (hereinafter referred to as HDL cholesterol) and LDL cholesterol, which are also important in the field of clinical diagnosis, and a reagent kit for use therein. The present invention further relates to a method for the continuous fractional determination of HDL cholesterol, LDL cholesterol and total cholesterol [the term is used to mean total cholesterol in HDL, LDL, very low density lipoproteins (hereinafter referred to as VLDL) and chylomicron (hereinafter referred to as CM)], which are important in the field of clinical diagnosis, as well as a reagent kit to be used therefor.
In general, HDL is called good cholesterol since HDL functions to remove cholesterol accumulated on arterial walls and transport cholesterol to liver. On the other hand, LDL is generally termed bad cholesterol because of its action to transport cholesterol to peripheral tissues including arterial walls. In the field of clinical investigations, the levels of HDL cholesterol, LDL cholesterol and total cholesterol are useful indices for total judgement of lipid-related diseases such as arteriosclerosis, etc.
These cholesterol levels are separately determined using reagents exclusively specific to each type of cholesterol so that an autoanalyzer is designed so as to be suitable for individual determination of these cholesterol levels. It has been desired to further improve the specificity of a reagent to each cholesterol. Besides, no simple and automated method for the continuous fractional determination of HDL cholesterol, LDL cholesterol, total cholesterol, etc in the same detection system is known.
An object of the present invention is to provide a method for the quantitative determination of LDL cholesterol and a determination reagent for use in such a method.
Another object of the present invention is to provide a method for the continuous fractional determination of HDL cholesterol and LDL cholesterol in the same sample and a reagent kit for use therein.
More specifically, the present invention relates to (1) through (27) below.
(1) A method for quantitatively determining LDL cholesterol in a biological sample, which comprises
performing the reaction of cholesterol in the presence of:
a) a biological sample,
b) cholesterol esterase and cholesterol oxidase or cholesterol dehydrogenase (hereinafter collectively referred to as CH enzymes), and
c) a reagent enabling the CH enzymes of b) to act only on LDL cholesterol, and
measuring the amount of the hydrogen peroxide or reduced coenzyme formed by the reaction to quantitatively determine the concentration of LDL cholesterol.
(2) The method according to (1), wherein the reagent enabling CH enzymes to act only on LDL cholesterol is a reagent containing at least a polyoxyethylene derivative and a polyoxyethylene-polyoxypropylene copolymer.
(3) The method according to (2), wherein the polyoxyethylene derivative is a polyoxyethylene alkyl ether or a polyoxyethylene alkylaryl ether.
(4) The method according to (2) or (3), wherein the polyoxyethylene-polyoxypropylene copolymer is a surfactant represented by general formula (I):
HOxe2x80x94(C2H4O)axe2x80x94(C3H6O)bxe2x80x94(C2H4O)cxe2x80x94Hxe2x80x83xe2x80x83(I)
(wherein a, b and c, which may be the same or different, each represents an integer of 1 to 200). 
(5) A method for the continuous fractional determination of HDL cholesterol and LDL cholesterol in a biological sample, which comprises
subjecting cholesterol to the first reaction in the presence of:
a) a biological sample,
b) CH enzymes, and
c) a reagent enabling the CH enzymes of b) to act only on HDL cholesterol, and
measuring the amount of the hydrogen peroxide or reduced coenzyme formed by the first reaction to quantitatively determine the concentration of HDL cholesterol,
then adding
d) a reagent enabling the CH enzymes of b) to act only on LDL cholesterol,
subjecting cholesterol to the second reaction, and
measuring the amount of the hydrogen peroxide or reduced coenzyme formed by the second reaction to quantitatively determine the concentration of LDL cholesterol.
(6) A method for the continuous fractional determination of HDL cholesterol and LDL cholesterol in a biological sample, which comprises
subjecting cholesterol to the first reaction in the presence of:
a) a biological sample,
b) CH enzymes, and
c) a reagent enabling the CH enzymes of b) to act only on HDL cholesterol, and
measuring the amount of the hydrogen peroxide or reduced coenzyme formed by the first reaction to quantitatively determine the concentration of HDL cholesterol,
then adding
d) CH enzymes, and
e) a reagent enabling the CH enzymes of d) to act only on LDL cholesterol,
subjecting cholesterol to the second reaction, and
measuring the amount of the hydrogen peroxide or reduced coenzyme formed by the second reaction to quantitatively determine the concentration of LDL cholesterol.
(7) The method according to (5) or (6), wherein the reagent enabling CH enzymes to act only on LDL cholesterol is a reagent containing at least a polyoxyethylene derivative and a polyoxyethylene-polyoxypropylene copolymer.
(8) The method according to (7), wherein the polyoxyethylene derivative is a polyoxyethylene alkyl ether or a polyoxyethylene alkylaryl ether.
(9) The method according to (7) or (8), wherein the polyoxyethylene-polyoxypropylene copolymer is a surfactant represented by general formula (I):
HOxe2x80x94(C2H4O)axe2x80x94(C3H6O)bxe2x80x94(C2H4O)cxe2x80x94Hxe2x80x83xe2x80x83(I)
(wherein a, b and c, which may be the same or different, each represents an integer of 1 to 200). 
(10) A method for the continuous fractional determination of HDL cholesterol and total cholesterol in a biological sample, which comprises
subjecting cholesterol to the first reaction in the presence of:
a) a biological sample,
b) CH enzymes, and
c) a reagent enabling CH enzymes of b) to act only on HDL cholesterol, and
measuring the amount of the hydrogen peroxide or reduced coenzyme formed by the first reaction to quantitatively determine the concentration of HDL cholesterol,
then adding
d) a reagent enabling the CH enzymes of b) to act on cholesterol in all lipoproteins,
subjecting cholesterol to the second reaction, and
measuring the amount of the hydrogen peroxide or reduced coenzyme formed by the second reaction to quantitatively determine the concentration of total cholesterol.
(11) A method for the continuous fractional determination of HDL cholesterol and total cholesterol in a biological sample, which comprises
subjecting cholesterol to the first reaction in the presence of:
a) a biological sample,
b) CH enzymes, and
c) a reagent enabling the CH enzymes of b) to act only on HDL cholesterol, and
measuring the amount of the hydrogen peroxide or reduced coenzyme formed by the first reaction to quantitatively determine the concentration of HDL cholesterol,
then adding
d) CH enzymes, and
e) a reagent enabling the CH enzymes of d) to act on cholesterol in all lipoproteins,
subjecting cholesterol to the second reaction, and
measuring the amount of the hydrogen peroxide or reduced coenzyme formed by the second reaction to quantitatively determine the total cholesterol.
(12) The method according to any one of (5) through (11), wherein the reagent enabling CH enzymes to act only on cholesterol in HDL is a reagent for aggregating lipoproteins other than HDL.
(13) The method according to (12), wherein the reagent for aggregating lipoproteins other than HDL further contains a nonionic surfactant that does not solubilize the aggregated lipoproteins.
(14) The method according to (12) or (13), wherein the reagent for aggregating lipoproteins other than HDL is a reagent comprising heparin or a salt thereof, phosphotungstic acid or a salt thereof, dextran sulfuric acid or a salt thereof, polyethylene glycol, sulfonated cyclodextrin or a salt thereof, sulfonated oligosaccharide or a salt thereof, or a mixture thereof and a divalent metal salt.
(15) The method according to (6) or (11), wherein the CH enzymes used in the first reaction of cholesterol are chemically modified enzymes and the CH enzymes used in the second reaction of cholesterol are enzymes that are not chemically modified.
(16) The method according to any one of (10) through (15), wherein the reagent enabling the CH enzymes to act on cholesterol in all lipoproteins is a reagent containing a lipoprotein solubilizing surfactant.
(17) A reagent for determining LDL cholesterol comprising CH enzymes and a reagent enabling the CH enzymes to act only on LDL cholesterol.
(18) The reagent for determining LDL cholesterol according to (17), wherein the reagent enabling the CH enzymes to act only on LDL cholesterol is a reagent containing at least a polyoxyethylene derivative and a polyoxyethylene-polyoxypropylene copolymer.
(19) The reagent according to (18), wherein the polyoxyethylene derivative is a polyoxyethylene alkylaryl ether.
(20) The reagent according to (18) or (19), wherein the polyoxyethylene-polyoxypropylene copolymer is a surfactant represented by general formula (I):
HOxe2x80x94(C2H4O)axe2x80x94(C3H6O)bxe2x80x94(C2H4O)cxe2x80x94Hxe2x80x83xe2x80x83(I)
(wherein a, b and c, which may be the same or different, each represents an integer of 1 to 200). 
(21) A reagent kit for the fractional determination of HDL cholesterol and LDL cholesterol comprising a first reagent and a second reagent, said first reagent comprising a reagent for aggregating lipoproteins other than HDL lipoprotein and a reagent containing CH enzymes, and said second reagent comprising a reagent enabling CH enzymes to act only on LDL cholesterol.
(22) The reagent kit according to (21), wherein the reagent enabling CH enzymes to act only on LDL cholesterol is a reagent containing a polyoxyethylene derivative and a polyoxyethylene-polyoxypropylene copolymer.
(23) The reagent kit according to (21), wherein the polyoxyethylene derivative is a polyoxyethylene alkylaryl ether.
(24) The reagent kit according to (21) or (22), wherein the polyoxyethylene-polyoxypropylene copolymer is a surfactant represented by general formula (I):
HOxe2x80x94(C2H4O)axe2x80x94(C3H6O)bxe2x80x94(C2H4O)cxe2x80x94Hxe2x80x83xe2x80x83(I)
(wherein a, b and c, which may be the same or different, each represents an integer of 1 to 200).
(25) A reagent kit for the fractional determination of HDL cholesterol and total cholesterol comprising a first reagent and a second reagent, said first reagent comprising a reagent for aggregating lipoproteins other than HDL lipoprotein and a reagent containing CH enzymes, and said second reagent comprising a reagent enabling CH enzymes to act on cholesterol in all lipoproteins.
(26) The reagent kit according to (25), wherein the reagent enabling CH enzymes to act on cholesterol in all lipoproteins further contains a lipoprotein solubilizing surfactant.
(27) The reagent kit according to any one of (21) through (26), wherein the reagent for aggregating lipoproteins other than HDL lipoprotein is a reagent comprising heparin or a salt thereof, phosphotungstic acid or a salt thereof, dextran sulfuric acid or a salt thereof, polyethylene glycol, sulfonated cyclodextrin or a salt thereof, sulfonated oligosaccharide or a salt thereof, or a mixture thereof and a divalent metal salt.
Hereinafter the present invention will be described in more detail.
As described above, the present invention relates to a method for the quantitative determination of LDL cholesterol which comprises adding to a biological sample containing various types of lipoproteins a specific reagent enabling CH enzymes to act only on LDL cholesterol (hereinafter referred to as reagent A) and a reagent for use in such a method.
As described above, the present invention also relates to a method for the fractional determination of HDL cholesterol and LDL cholesterol which comprises adding to a biological sample containing various types of lipoproteins a specific reagent enabling CH enzymes to act only on HDL cholesterol (hereinafter referred to as reagent B) to quantitatively determine HDL cholesterol and then adding reagent A to quantitatively determine LDL cholesterol as well as a reagent kit for use therein.
As described above, the present invention further relates to a method for the fractional determination of HDL cholesterol and total cholesterol which comprises adding to a biological sample containing various types of lipoproteins the reagent B to quantitatively determine HDL cholesterol and then adding to the reaction mixture a reagent enabling CH enzymes to act on cholesterol in all lipoproteins (hereinafter referred to as reagent C) to quantitatively determine total cholesterol as well as a reagent kit for use therein.
A biological sample to which the present invention is to be applied is not particularly limited. More specifically, blood itself or blood fractions such as plasma or serum, etc. may be used as the sample.
The reactions for quantitatively determining cholesterol in the present invention are generally carried out in an aqueous medium, preferably in a buffer solution.
Buffers useful in the buffer solution include tris(hydroxymethyl)aminomethane, phosphate buffer, borate buffer and Good""s buffer. Examples of Good""s buffer are N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), N-(2-acetamido)iminodiacetic acid (ADA), N, N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 3-[N,N-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (DIPSO), N-(2-hydroxyethyl)piperazine-Nxe2x80x2-2-ethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonci acid (hereinafter referred to as MES), 3-(N-morpholino)propanesulfonic acid (hereinafter referred to as MOPS), 3-(N-morpholino)-2-hydroxypropanesulfonic acid (MOPSO), piperazine-N,Nxe2x80x2-bis(2-ethanesulfonic acid) (hereinafter referred to as PIPES), piperazine-N,Nxe2x80x2-bis(2-hydroxypropane-3-sulfonic acid) (POPSO), etc.
The pH of the buffer solution is 5 to 10, preferably 6 to 9. The concentration of the buffer to be used is 5 to 500 mM, preferably 20 to 200 mM.
The reagent A which enables CH enzymes to act only on LDL cholesterol is a reagent that does not enable CH enzymes to act on cholesterol in HDL, VLDL and CM. The reagent A also enables CH enzymes to act only on LDL cholesterol even in the presence of a reagent for aggregating lipoproteins other than HDL, which will be later described.
The reagent A is typically a reagent containing at least a polyoxyethylene derivative and a polyoxyethylene-polyoxypropylene copolymer.
Suitable polyoxyethylene derivative is exemplified by a polyoxyethylene alkylaryl ether, a polyoxyethylene alkyl ether, etc., having the alkyl moiety of at least 8 carbon atoms, e.g., octyl, nonyl, etc. and having the aryl moiety being phenyl, etc.
Specific examples of the polyoxyethylene derivative include commercially available Nonion HS-210, Nonion HS-215, Nonion NS-208.5 and Nonion HS-208 (all produced by NOF Corporation) and Emulgen L-40, Emulgen 911 and Emulgen 810 (all produced by Kao Corporation). The hydrophile-lipophile balance (hereinafter referred to as HLB) of the polyoxyethylene derivative is preferably 9 to 20.
The polyoxyethylene-polyoxypropylene copolymers may be either random copolymers or block copolymers of polyoxyethylene and polyoxypropylene. An example of the copolymer is a compound represented by general formula (I):
HOxe2x80x94(C2H4O)axe2x80x94(C3H6O)bxe2x80x94(C2H4O)cxe2x80x94Hxe2x80x83xe2x80x83(I)
(wherein a, b and c, which may be the same or different, each represents an integer of 1 to 200). 
Examples of the compounds represented by general formula (I) include commercially available compounds such as Pluronic L-121, Pluronic L-122, Pluronic L-101, Pluronic P-103 and Pluronic F-108 (all produced by Asahi Denka Kogyo K. K.). The molecular weight of the polypropylene glycol moiety in the compounds of general formula (I) is preferably at least 2,050, more preferably 2,750 or more, most preferably 3,250 or more. The HLB of the polyoxyethylene-polyoxypropylene copolymers is in preferably 1 to 6.
The respective concentration of the polyoxyethylene derivatives and polyoxyethylene-polyoxypropylene copolymers used is not specifically limited but is preferably 0.001 to 10%, more preferably 0.01 to 5%, most preferably 0.05 to 1%.
Examples of the reagent B that enables CH enzymes to act only on HDL cholesterol are reagents for aggregating lipoproteins other than HDL and antibodies to lipoproteins other than HDL.
Reagents for aggregating lipoproteins other than HDL are generally those containing agents for aggregating these lipoproteins and/or divalent metal salts. Examples of the aggregating agent include heparin or salts thereof, phosphotungstic acid or salts thereof, dextran sulfuric acid or salts thereof, polyethylene glycol, sulfated cyclodextrin or salts thereof, sulfated oligosaccharide or salts thereof, and mixtures thereof. Examples of the cyclodextrin include xcex1-cyclodextrin, xcex2-cyclodextrin and xcex3-cyclodextrin. Examples of the oligosaccharide include maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose. Examples of the salts include sodium, potassium, lithium, ammonium and magnesium salts. Examples of the divalent metal salt include magnesium, calcium, manganese and nickel salts.
Preferable examples of the aggregating agent used include 0.02 to 10 mM heparin having a molecular weight of 5, 000 to 20, 000 or salts thereof, 0.1 to 10 mM phosphotungstic acid having a molecular weight of 4,000 to 8,000 or salts thereof, 0.01 to 5 mM dextran sulfuric acid having a molecular weight of 10,000 to 500,000 or salts thereof, 0.1 to 20 mM dextran sulfuric acid having a molecular weight of 1,000 to 10,000 or salts thereof, 0.3 to 100 mM polyethylene glycol (PEG) having a molecular weight of 4,000 to 25,000, 0.1 to 50 mM sulfated cyclodextrin having a molecular weight of 1,000 to 3,000 or salts thereof, 0.1 to 50 mM sulfated oligosaccharide having a molecular weight of 400 to 3,000 or salts thereof, and mixtures thereof. More preferred examples are 0.03 to 1 mM heparin having a molecular weight of 14,000 to 16,000 or salts thereof, 0.1 to 3 mM phosphotungstic acid having a molecular weight of 5,000 to 7,000 or salts thereof, 0.01 to 5 mM dextran sulfuric acid having a molecular weight of 150,000 to 250,000 or salts thereof, 0.1 to 10 mM dextran sulfuric acid having a molecular weight of 1,000 to 5,000 or salts thereof, 1.0 to 50 mM PEG having a molecular weight of 5,000 to 22,000, 0.1 to 10 mM sulfated cyclodextrin having a molecular weight of 1,000 to 2,000 or salts thereof, 0.1 to 10 mM sulfated oligosaccharide having a molecular weight of 400 to 2,000 or salts thereof, and mixtures thereof.
Preferred examples of the divalent metal salt include the salts of magnesium, calcium, manganese, nickel and cobalt, the concentration of which is 0.1 to 50 mM. Preferably, the magnesium salt is used in a concentration of 0.1 to 50 mM.
It is preferred that the agents for aggregating lipoproteins other than HDL further contain a nonionic surfactant that does not dissolve the aggregated lipoproteins.
Examples of the nonionic surfactant that does not dissolve the aggregated lipoproteins include a polyoxyethylene alkyl ether, a polyoxyethylene alkylaryl ether, a polyoxyethylene-polyoxypropylene copolymer, a polyoxyethylene alkyl ether sulfuric acid salts and an alkylbenzene sulfonate. Among these surfactants, polyoxyethylene ethers [Emulgen 220 (Kao Corporation), etc.] are particularly desired as the polyoxyethylene alkyl ether; commercially available Emulgen 66, etc. as the polyoxyethylene alkyl aryl ether; commercially available Pluronic F88 (Asahi Denka Kogyo K. K.) as the polyoxyethylene-polyoxypropylene condensate, commercially available Emal 20C (Kao Corporation) as the polyoxyethylene alkyl ether sodium sulfate, and sodium dodecyl benzenesulfonate as the alkyl benzenesulfonic acid salt.
The nonionic surfactant that does not dissolve the aggregated lipoproteins can be used in combination, so long as the surfactant does not enable CH enzymes to act on LDL cholesterol. However, it is preferable to use the nonionic surfactant solely. The concentration of the nonionic surfactant is not particularly limited but is preferably 0.01 to 10%, more preferably 0.1 to 5%.
Examples of the antibodies to lipoproteins other than HDL include an antiapo-lipoprotein B antibody, an antiapo-lipoprotein C antibody, an antiapo-lipoprotein E antibody and an anti-xcex2-lipoprotein antibody. These antibodies may be employed solely or in combination. The antibodies may be either polyclonal or monoclonal. The antibodies may also be chemically or enzymatically degraded or modified.
As the reagent C enabling CH enzymes to act on cholesterol in all lipoproteins, there are, for example, surfactants that dissolve all lipoproteins.
As the surfactants above, there are used nonionic surfactants that dissolve HDL, LDL, VLDL and CM. Specific examples of such surfactants are nonionic surfactants commercially available as Triton X-100, polyoxyethylene alkyl ethers such as Emulgen 106, Emulgen 108, Emulgen 709, etc. These surfactants may be used solely or in combination. The concentration of the surfactants is not particularly limited but is preferably 0.01 to 10%, more preferably 0.1 to 5%.
As the enzymes having the activities of cholesterol esterase, cholesterol oxidase and cholesterol dehydrogenase which may be used in the present invention, there are, for example, cholesterol esterase and lipoprotein lipase derived from microorganisms or animals having the ability to hydrolyze cholesterol ester, cholesterol oxidase derived from microorganisms having the ability to oxidize cholesterol to produce hydrogen peroxide, and cholesterol dehydrogenase derived from microorganisms or animals.
These enzymes can be employed depending upon specificity to substrate. In the case of the quantitative determination of HDL cholesterol, it is preferred to use an enzyme specific to the cholesterol and for the quantitative determination of LDL cholesterol, an enzyme specific thereto is preferably used. In order to further improve the specificity and stability of these enzymes, enzymes that are chemically modified with a group having polyethylene glycol as a main component, a water-soluble oligosaccharide residue, or a sulfopropyl group may also be used. Furthermore, enzymes obtained by genetic engineering may also be used.
Examples of the reagent for modifying the enzymes (chemical modifier) include compounds wherein polyethylene glycol and a group capable of bonding to an amino group are connected, e.g. Sun Bright VFM4101 (NOF Corporation) wherein polyethylene glycol and a group capable of bonding to an amino group such as N-hydroxysuccinimido group are connected, Sun Bright AKM series, ADM series and ACM series [all manufactured by NOF Corporation, Chemical Engineering Monographs (Kagaku Kogaku Ronbunshu), 20 (3), 459 (1994)] which are compounds having the polyalkylene glycol structure and the acid anhydride structure, compounds wherein a polyethylene glycol-polypropylene glycol copolymer and a group capable of bonding to an amino group are connected, copolymers of polyethylene glycol monomethacryl monomethyl ether and maleic anhydride, etc. Furthermore, activated polyurethane P4000 (Boehringer Mannheim, Directions for Enzyme Modification Set) which is a polyurethane chemical modifier, Dextran T40, which is a dextran chemical modifier, and activated TCT (Boehringer Mannheim, Directions for Enzyme Modification Set), 1,3-propanesultone, etc. may also be used. By the use of these chemical modifiers, the enzymes can be modified with a group having polyethylene glycol as a main component, a group having polypropylene glycol as a main component, a group having a copolymer of polypropylene glycol and polyethylene glycol, a group containing a saccharide in the structure, a sulfopropyl group, a polyurethane group, etc.
A method for the reaction of an enzyme with the above chemical modifier is described in Yuji Inada, xe2x80x9cTanpakushitu-no-Hybrid (Hybrid of Proteins)xe2x80x9d published by Kyoritsu Publishing Co. (1987), etc. Typically, when using, e.g., Sun Bright, the enzyme is dissolved in a buffer solution such as HEPES buffer of pH 8 or above, then, e.g., 0.01-500-fold molar amount of Sun Bright is added to the solution at 0xc2x0 C. to 50xc2x0 C., followed by stirring for 5 to 60 minutes. The resulting reaction mixture is used as it is, or if necessary, after removal of low molecular weight compounds with ultrafilter.
According to the present invention, the cholesterol esterase, cholesterol oxidase and cholesterol dehydrogenase are preferably used in the reaction mixture at a concentration of 0.01 to 200 U/ml, more preferably 0.1-100 U/ml.
In the present invention, the CH enzymes used to quantitatively determine HDL cholesterol may be used as they are for quantitative determination of LDL cholesterol or cholesterol other than HDL or total cholesterol.
Alternatively, for the quantitative determination of LDL cholesterol or cholesterol other than HDL or total cholesterol, CH enzymes having the same or different specificities may be newly added to the system.
Preferably, chemically modified CH enzymes are used for the quantitative determination of HDL cholesterol and for the determination of cholesterol in lipoproteins other than LDL or HDL or total cholesterol, CH enzymes without any chemical modification are used.
In the reaction of cholesterol according to the present invention, a surfactant or cholic acid which is conventionally used to activate CH enzymes may also be employed as far as they do not affect the reaction specificity. Further, various salts for solubilizing proteins such as globulin may also be used.
As the surfactant for activating the CH enzymes, anionic surfactants are used, e.g., at a concentration of 0 to 1%. Examples of the cholic acid are cholic acid, deoxycholic acid, taurocholic acid and chenodeoxycholic acid. The cholic acid is used at a concentration of 0 to 5%. Examples of the anionic surfactant include an alkyl sulfonate such as 1-pentasulfonate, 1-hexasulfonate, 1-heptasulfonate and1-octasulfonate. These surfactants are used at a concentration of 0 to 5%.
Examples of the salts include sodium chloride, sodium sulfate, potassium chloride, potassium sulfate, magnesium chloride, magnesium sulfate, magnesium acetate, lithium chloride, lithium sulfate, ammonium chloride, ammonium sulfate, magnesium nitrate and calcium nitrate. These salts are used at a concentration of 0 to 100 mM.
When the reaction of cholesterol is carried out with cholesterol esterase and cholesterol oxidase, hydrogen peroxide is formed. The formed hydrogen peroxide can be quantitatively determined, using e.g. 4-aminoantipyrine and a phenol, 4-aminoantipyrine and Trinder""s reagent, or a highly sensitive chromogen in the presence of peroxidase.
Examples of phenols are phenol, 4-chlorophenol, m-cresol and 3-hydroxy-2,4,6-triiodobenzoic acid (HTIB).
Examples of the Trinder""s reagents (General Catalog of Dojin Kagaku Kenkyusho, 19th ed., 1994) are anilines such as N-sulfopropylaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (TOOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline (MAOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS), N-ethyl-N-sulfopropyl-m-toluidine (TOPS), N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS), N,N-dimethyl-m-toluidine, N,N-disulfopropyl-3,5-dimethoxyaniline, N-ethyl-N-sulfopropyl-m-anisidine, N-ethyl-N-sulfopropylaniline, N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline, N-sulfopropyl-3,5-dimethoxyaniline, N-ethyl-N-sulfopropyl-3,5-dimethylaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-anisidine, N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline and N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(3-methylphenyl)-Nxe2x80x2-succinylethylenediamine (EMSE), and N-ethyl-N-(3-methylphenyl)-Nxe2x80x2-acetylethylenediamine.
As the highly sensitive chromogen, there are 10-(N-methylcarbamoyl)-3,7-bis(dimethylamino)phenothiadine (MCDP) disclosed in Japanese Published Examined Patent Application No. 33479/85, bis[3-bis(4-chlorophenyl)methyl-4-dimethylaminophenyl]amine (BCMA) disclosed in Japanese Published Examined Patent Application No. 27839/92, the compounds disclosed in Japanese Published Unexamined Patent Application No. 296/87, etc.
The chromogen is preferably used in a concentration of 0.01 to 10 mg/ml.
When the reaction of cholesterol is carried out with cholesterol esterase and cholesterol dehydrogenase in the presence of an oxidized coenzyme, NAD(P), as a substrate, a reduced coenzyme, NAD(P)H, is formed. The formed NAD(P)H can be quantitatively determined by measuring the absorbance of a reaction mixture at 300 to 500 nm, preferably 330 to 400 nm, particularly preferably about 340 nm. The determination of NAD(P)H may otherwise be made by forming a formazan pigment through addition of diaphorase and a tetrazolium salt and then measuring the formazan pigment by colorimetry.
The reaction for the quantitative determination of LDL cholesterol is carried out at 10 to 50xc2x0 C., preferably 30 to 40xc2x0 C., usually at 37xc2x0 C., for 1 to 30 minutes, preferably 2 to 10 minutes.
In the fractional determination, the reaction for quantitatively determining HDL cholesterol (hereinafter referred to as the first reaction) are carried out at 10 to 50xc2x0 C., preferably 30 to 40xc2x0 C., usually at 37xc2x0 C., for 1 to 30 minutes, preferably 2 to 10 minutes; the reactions for quantitatively determining LDL cholesterol or total cholesterol (hereinafter referred to as the second reaction) are carried out at 10 to 50xc2x0 C., preferably 30 to 40xc2x0 C., usually at 37xc2x0 C., for 1 to 30 minutes, preferably 2 to 10 minutes. The start of the second reaction may be at any stage, e.g., after the first reaction is substantially completed or during the first reaction, so long as the quantitative determination of HDL is completed. The second reaction is initiated by adding the reagent A enabling the CH enzymes to act specifically on LDL cholesterol or the reagent C enabling the CH enzymes to act on cholesterol in all lipoproteins and, if necessary, CH enzymes. The hydrogen peroxide or reduced coenzyme [NAD(P)H] thus formed by the second reaction is quantitatively determined using the same reagents as used in the first reaction as they are, or, if necessary and desired, reagents may be newly added to the system.
In the present invention where HDL cholesterol and LDL cholesterol are fractionally determined by first performing the reaction of HDL cholesterol followed by the reaction of LDL cholesterol, the reaction of LDL cholesterol is initiated by adding the reagent A as described above. In this case, when the first reaction of HDL cholesterol is carried out by adding the nonionic surfactant that does not dissolve the aggregated lipoproteins other than HDL, i.e., by adding either the polyoxyethylene derivative or the polyoxyethylene-polyoxypropylene copolymer, the second reaction of LDL cholesterol may also be initiated by adding such a surfactant as forming the reagent A in combination with the surfactant used in the reaction of HDL cholesterol.
The concentration of cholesterol in each lipoprotein is calculated by the following equation based on a difference in absorbance (xcex94OD) before and after each reaction using a test sample and a difference in absorbance (xcex94ODstd) using a sample with a known concentration of cholesterol in various lipoproteins.
The concentration of LDL cholesterol can be determined by the following equation:
xcex94OD÷xcex94ODstdxc3x97(known concentration of LDL cholesterol)
The concentration of HDL cholesterol can be determined by, e.g., the following equation:
xcex94OD÷xcex94ODstdxc3x97(known concentration of HDL cholesterol)
In the fractional determination, when the compounds formed in the first and second reactions are the same and they are detected by the same method, the concentration of total cholesterol can be calculated according to the following equation, using the difference in absorbance before the first reaction and after the second reaction:
xcex94OD÷xcex94ODstdxc3x97(known concentration of total cholesterol)
The reagent of the present invention for quantitatively determining LDL cholesterol comprises CH enzymes and a reagent comprising the polyoxyethylene derivative and the polyoxyethylene-polyoxypropylene copolymer. The above reagent for quantitatively determining LDL cholesterol may further contain, if necessary, the aforesaid buffers, reagents for aggregating lipoproteins other than HDL, surfactants used for quantitatively determining cholesterol, cholic acids, various salts, enzymes such as peroxidase, chromogens such as 4-aminoantipyrine and Trinder""s reagents or oxidized coenzymes such as NAD(P).
The reagent kit of the present invention for the fractional determination of HDL cholesterol and LDL cholesterol comprises a first reagent and a second reagent. For example, the first reagent comprises a reagent containing an aggregating agent for lipoproteins other than HDL and CH enzymes and the second reagent comprises a reagent containing the polyoxyethylene derivative and the polyoxyethylene-polyoxypropylene copolymer.
The reagent kit of the present invention for the fractional determination of HDL cholesterol and total cholesterol comprises a first reagent and a second reagent. For example, the first reagent comprises a reagent containing an aggregating agent for lipoproteins other than HDL and CH enzymes and the second reagent comprises a reagent containing a nonionic surfactant that dissolves all lipoproteins (HDL, LDL, VLDL and CM).
The first and second reagents of the reagent kit in accordance with the present invention may further contain, if necessary and desired, the aforesaid buffers, surfactants used for the quantitative determination of cholesterol, cholic acids, various salts, enzymes such as peroxidase, chromogens such as 4-aminoantipyrine and Trinder""s reagents, oxidized coenzymes such as NAD(P).
In the second reagent, the source of CH enzymes may be the same as or different from the first reagent. It is preferred that the chemically modified enzyme described above is used as the CH enzyme for the first reagent and a CH enzyme not chemically modified is used as the CH enzyme for the second reagent.